Direct measurement of collagenase in colonic anastomosis

Collagenase has been implicated in colonic anastomotic dehiscence but the enzyme has not previously been specifically measured in colonic healing. A 72 h tissue culture method for colonic tissue and a radio chemical assay for collagenase were adapted to measure the enzyme in healing rabbit colon, with specificity of the assay confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis. Normal and postoperative colon secreted collagenase, predominantly in a latent form, in the first 24 h of culture. Total activity reached a plateau after 48 and 72 h in culture, when 50–70 per cent of the enzyme was in an active form. At these times in culture, activity was significantly higher than after 24 h (P< 0.001). One day after anastomosis the total amount of collagenase secreted in culture was higher than normal but the increase did not achieve significance. Three days after anastomosis the colon secreted more collagenase than explants from 1 day postoperative tissue (P<0.002). The proportion of active enzyme in the first 24 h in culture was also increased. Since active collagenase can be measured in culture medium from both normal and postoperative colon, the tissue may be secreting plasminogen activator which allows plasmin to activate the enzyme. The increase in collagenase after operation coincided with a decrease in collagen concentration in the colon wall, measured by hydroxyproline. This supports previous suggestions that collagenase contributes to anastomotic dehiscence. However, the findings must be interpreted with caution as the variance of the results was shown to be predominantly due to time in culture, suggesting this could be a bigger influence than the operation itself. In addition, our previously reported immunohistochemical study of this system indicated that collagenase only occurred in a localized region, restricted to the everted portion of the anastomosis, with the activity being tightly controlled by its inhibitor, tissue inhibitor of metalloproteinases.